1. Field of the Invention
The present invention provides antisense oligodeoxynucleotides targeted to exon sequences flanking donor splice sites which regulate expression of TNF-.alpha..
2. Description of Related Art
There has been increasing interest in the development of antisense oligodeoxyribonucleotides (AS-ODNs hereinafter) as therapeutic agents and experimental tools (Stein and Cheng, 1993; Wagner, 1994). However, despite the improvement in affinity for target RNA, increased resistance to nucleolytic cleavage, and enhanced delivery of AS-ODNs to cells and their nuclei (Hodges and Crooke, 1995), high concentrations of AS-ODNs continue to be required to inhibit gene expression. To some extent, high AS-ODN concentrations have hampered the development of this technology as an effective pharmacological agent because of cost and non-specific AS-ODN actions.
Many genes encode pre-mRNAs containing introns that are removed by a splicing process that is directed by a complex of small nuclear ribonucleic proteins (snRNPs) called the spliceosome (Staley and Guthrie, 1998). Several reports indicate that gene expression is effectively inhibited by AS-ODNs targeting the intron/exon boundaries of splice sites (Boeve and De Ley, 1994; Dominski and Kole, 1996; Dominski and Kole, 1994; Hodges and Crooke, 1995; Moulds et al., 1995), likely because these domains direct splicing events (Staley and Guthrie, 1998). It has previously been shown in cell free systems that the degree of sequence variability at splice sites influences splicing events (Dominski and Kole, 1994), suggesting that pre-mRNAs with variant splice site sequences would be ideal targets for AS-ODN treatment (Hodges and Crooke, 1995). Since exon sequences upstream of donor (5'), and downstream of acceptor (3') splice sites within pre-mRNA play a critical role in processing RNA (Staley and Guthrie, 1998), it is plausible that these sites encode RNA domains highly susceptible to AS-ODN-mediated inhibition of gene expression. To date, this hypothesis has not been tested systematically in a biologically relevant system such as tumor necrosis factor alpha (TNF-.alpha.) production in cell culture or in vivo.
Under normal conditions, TNF-.alpha.'s expression is tightly regulated by rapid mRNA turnover (Gearing et al., 1995). However, in disease states, its expression is perturbed, resulting in overexpression (Sharief and Hentges, 1991; Tracey and Cerami, 1994). TNF-.alpha. is implicated in the pathogenesis of several inflammatory diseases including multiple sclerosis (MS) (French-Constant, 1994), rheumatoid arthritis (RA) (Lupia et al., 1996), viral infections such as human immunodeficiency virus (HIV) (Fauci, 1996) and, bacterial infections causing sepsis (Tomioka et al., 1996). TNF-.alpha. neutralizing antibodies (Givner et al., 1995), soluble TNF-.alpha. receptors (Moreland et al., 1997), or gene knockouts of the TNF receptor (p55) (Pfeffer et al., 1993) mitigate the harmful effects of TNF-.alpha. observed in several animal models of inflammation (Probert et al., 1995; Selmaj et al., 1991). However, these approaches do not limit TNF-.alpha. synthesis.
Several studies show that AS-ODNs targeting TNF-.alpha. mRNA limit TNF-.alpha. synthesis (Hartmann et al., 1996; Lefebvre d'Hellencourt et al., 1996; Rojanasakul et al., 1997; Taylor et al., 1996). However, in these reports, concentrations of AS-ODNs in excess of 2 .mu.M, were required to achieve significant inhibition. High concentrations of AS-ODNs may induce non-specific inflammatory cell responses (Hartmann et al., 1996) as well as other non-specific effects (Gao et al., 1992; Khaled et al., 1996; Perez et al., 1994). Nevertheless, earlier reports suggest that expression of other genes can be regulated by low concentrations (.ltoreq.1 .mu.M) of AS-ODNs (Hanecak et al., 1996; Miraglia et al., 1996). Therefore it would be useful to develop AS-ODNs that can be used in low concentrations to regulate TNF-.alpha. production in inflammatory responses.